Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

Transformation Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your destination vector (typically 100 µg/ml ampicillin).

Expected results An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.

One tube format

If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway entry clone.

Mix well by vortexing briefly and incubate at 25°C for 4 hours. Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for large plasmids (=10 kb) and PCR products (=5 kb).

Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).